Microbial contamination has severe consequences for human and environmental health, not only due to its effect on health care and prevention, but also due to its long-reaching financial impact (Hutton G, Bartram J, “Global costs of attaining the Millennium Development Goal for water supply and sanitation”, Bulletin of the World Health Organization, 86(1): 13-9, 2008). Bacteria, viruses, yeasts and protozoa are causal agents of an extraordinary number of diseases.
These infectious microorganisms are biological agents which reach their hosts through supports serving as a carrier, they are established in the body of their host and cause damage. According to the present invention, a microorganism is a prokaryotic or eukaryotic microscopic organism, not including viruses.
Bacteria fundamentally include those known as pathogenic bacteria, such as, for example, species of Enterobacteriaceae, Vibrionaceae, Bacillus, Escherichia, Streptococcus, Pseudomonas, Salmonella, Legionella, Enterobacter, etc.
Bacteria can spread to vertebrates by different methods, specifically through foods (for example, Salmonella), contaminated beverages (for example, Escherichia coli) or in droplets which can be transmitted by air (for example, Legionella). Thus, their absence in places such as cooling towers, water conduits in hospitals and hotels, public buildings such as schools, swimming pools, gymnasiums, resorts, spas, and the like, must be regularly checked. The process of the present invention will generally be applied to pathogenic bacteria.
These supports can contain the infectious microorganism, which causes the damage or disease, for a time sufficient to allow even the proliferation thereof, such that in only a few hours or days an infective concentration can be reached, above which it is highly likely that the host will be reached and damage will be caused.
It is obvious that it is necessary to detect the presence and concentration of the causal biological agent in these supports, which serve as a carrier for it in order to reach the host, for the purpose of establishing a correct prevention of the associated environmental and health risks. In particular, the rapid obtaining of results and the simplicity of the process for determining the presence and concentration thereof enables two fundamental issues for defining an efficient prevention and control strategy: 1) increasing the analysis frequency with a low and affordable cost facilitating the industrial application thereof; and 2) performing the analysis in situ in a short time, to prevent infective concentrations sustained over time and minimize the probability of the diseases, allowing appropriately applying corrective measures. This enables the integration of the analysis in routine monitoring and control operations of the risk environments.
The traditional methods for the detection and enumeration of microorganisms are slow and complex, therefore they require qualified personnel for performing several steps of handling (Noble R T, Weisberg S B. “A review of technologies for rapid detection of bacterium in recreational waters”. Journal of Water and Health, 3(4):381-92, 2005; Gracias K S., McKillip J L., “A review of conventional detection and enumeration processes for pathogenic bacterium in food”, Canadian Journal of Microbiology, 50(11):883-90, 2004; Rompré A., Serváis P., Baudarts J., de-Roubin M R., Laurent P., “Detection and enumeration of coliforms in drinking water: current processes and emerging approaches”, Journal of Microbiological Processes, 49(1):31-54, 2002). The standard method comprises isolating and counting the colonies of the bacterium grown under certain culture conditions. It also has serious limitations for performing a correct prevention of the risk associated with the microorganism, among others, the following: 1) the concentration of the microorganism which is to be determined in the samples can be low, and can furthermore be accompanied by other different microorganisms (microbiota). Consequently, it may be necessary to separate the microbiota before inoculating the culture media. Otherwise, said microbiota can compete favorably in the culture medium, and proliferate until masking or preventing the growth of the microorganism which is to be determined; and 2) the time required from the sampling until obtaining the count is generally greater than the time necessary for the microorganism to be duplicated, by several hours, and in some cases even by several days or weeks. This time is even significantly greater than the time which the microorganism may require to reach an infective concentration. Therefore, it is necessary to be able to detect the microorganism in the sample in a short time, for example approximately in 1 hour.
These limitations have promoted the development of other alternative methods. In particular, the use of recognition biomolecules, such as antibodies, antigens and nucleic acids, immobilized on a wide variety of supports (solid phase), is very interesting for the development of immunoassays in the environmental, food, biomedical, industrial and analytical chemistry field.
Some methods have been developed to substitute traditional techniques, such as, for example, immunoassays and PCR (polymerase chain reaction) techniques, which in addition to being slightly more rapid, are quite specific and sensitive.
Immunoassays, based on the antigen-antibody reaction, have recently been developed, which are commonly used to detect disease-causing pathogenic microorganisms (Meer R R, Park D L., “Immunochemical detection methods for Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in foods”, Reviews of environmental contamination and toxicology, 142:1-12, 1995). However, these methods have serious drawbacks. The following stand out among such drawbacks:
1) A negative result does not rule out the presence of the microorganism in the analyzed sample, since the microorganism could be at concentrations lower than the limit of detection of the method. Therefore, in immunoassays, including those of the ELISA (enzyme-linked immunosorbent assay) type, a minimum concentration of 105-106 cells is required for the detection of the microorganism, in a limited assay volume generally between 0.1 and 1.0 ml. This limit is highly conditioned because these methods do not allow using large volumes of sample since many commercially available immunoassays require a minimum concentration of cells of the microorganism for the detection, which makes it necessary to pre-enrich the sample and consequently significantly increase the assay time, necessary to reach a sufficient cell concentration of the microorganism which is to be determined.
2) Furthermore, the method does not distinguish between live or dead bacteria, because the free antigen is also detected, and because after the application of biocidal treatments false positives could be obtained due to the presence of dead bacteria or of the free antigen.
3) The limit of detection of the assay depends to a great extent on the microbioligical and chemical composition of the sample, because the presence of certain chemical compounds or of enzymatic activities of other microorganisms or even of the microorganism which is to be analyzed itself, can interfere in the detection and quantification of the latter.
Another set of techniques, the mentioned PCR techniques, are based on the amplification of a specific fragment of the genome of the microorganism. The nucleic acids of a sample are extracted and purified, to then be enzymatically amplified (by means of the polymerase enzyme) in cycles, and developed by means of electrophoresis or labeling with fluorescent probes. The main limitations of these methods are the high variability shown by the results depending on the matrix analyzed (Yaradou D F, Hallier-Soulier S, Moreau S, Poty F, Hillion Y, Reyrolle M, André J, Festoc G, Delabre K, Vandenesch F, Etienne J, Jarraud S. “Integrated real-time PCR for detection and monitoring of Legionella pneumophila in water systems”, Applied Environmental Microbiology, 73 (5): 1452-6, 2007; Joly P, Falconnet P A, André J, Weill N, Reyrolle M, Vandenesch F, Maurin M, Etienne J, Jarraud S., “Quantitative real-time Legionella PCR for environmental water samples: data interpretation”, Applied Environmental Microbiology, 72 (4):2801-8, 2006), and on the process for preparing the sample before the extraction of the nucleic acids, and in addition, on the large variety of inhibitors of the polymerase enzyme, which may be present in the samples.
A patent document representative of the analysis of bacteria in liquid samples by means of PCR is WO 01/40505 A1. Said document describes an analysis process for the presence of Legionella with an immunocapture step, and mentions that the main advantage of detecting Legionella by PCR is that it needs 24 to 48 h if the analysis is carried out by this method, in comparison with the traditional method of culture which needs 10 to 15 days to obtain the results. In said document, the bacteria can be captured by means of supports activated with antibodies, and it mentions the possibility of using magnetic beads, to then break the cells and extract the DNA, for the purpose of performing a PCR. It is therefore a method which requires breaking the integrity of the microorganism and which is subject to the known drawbacks of the PCR. The invention also relates to a kit for carrying out the method.
There is currently no universally accepted method for the preparation of the sample which allows obtaining reproducible results in all types of samples by means of the PCR technique, therefore it is necessary to continue developing new methods for the removal of inhibitors of the reaction and which simultaneously allows an efficient recovery of the microorganism.
Another option is the immunocapture of the microorganism which is to be determined, by means of using paramagnetic particles or spheres coated with antibodies directed against antigens of the microorganism in question. These immunomagnetic particles are mixed with the sample, form immunocomplexes with the specific microorganism, and allow separating and concentrating the captured microorganisms by means of applying a magnetic field, removing other components from the sample which can interfere with the determination.
For example, the use of an immunomagnetic separation by means of superparamagnetic particles or spheres coated with antibodies directed against antigens of the microorganism of interest, in combination with the real-time PCR technique, has been described (Yáñez M A, Carrasco-Serrano C, Barbera V M, Catalán V. “Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene”, Applied Environmental Microbiology, 71 (7):3433-41, 2005), but the recovery rate of the microorganism and the reproducibility of the capture thereof decrease with the increase of the complexity of the analyzed water.
The techniques for increasing the sensitivity of immunosorption assays have been focused on increasing the efficiency of the transduction of the signal, by means of using more efficient reading molecules and better detectors (L. J. Kricka, “Selected strategies for improving sensitivity and reliability of immunoassays”, Clinical Chemistry, Vol 40, 347-357, 1994). These techniques have involved the reduction of the pre-enrichment time of the sample, although not below 8 hours, and the limit of detection is at 106 cfu/l. For example, in order to increase the sensitivity of the method for the immunocapture of the microorganisms, several methods are applied, such as document US 2005/0202518 A1, for example, which applies immunomagnetic microspheres in the immunocapture step, but after a culture pre-enrichment step for 8-15 hours.
Patent document US 2006/0246535 A1 describes the detection of microorganisms in solution or dispersion, without pre-enrichment, using latex microspheres coated with antibodies, subsequently detecting the microorganism by means of measuring the agglutination.
Document ES 2 237 272 A1 describes a process for detecting and quantifying antibodies specific for Legionella pneumophila in sexological samples, by means of the agglutination-sedimentation of latex particles sensitized with an antigen of L. pneumophila. It also describes the method for obtaining sensitized latex particles and the reaction buffer in which the immunoreaction takes place.
Some patents intend to increase the sensitivity of the detection of microorganisms by means of using magnetic nanoparticles versus magnetic microparticles (1 μm=1000 nm). For example, patent US 2006/292555 A1 indicates that “there are, to date, no general and satisfactory assays that can detect bacteria at concentrations of <102 colony forming units per milliliter (cfu/ml) without pre-enriching the bacteria via a culture process”. Explicitly, the mentioned patent states that the sensitivity achieved, of the order of 10-100 bacteria/ml, cannot be achieved by means of microparticles, understanding as such those the diameter of which is in the order of one micron, not of one nanometer. Thus, said patent document describes a method for detecting pathogens which comprises using magnetic nanoparticles formed by an antibiotic, the vancomycin bound to the surface of FePt (iron-platinum) nanoparticles.
There are several documents the method of which comprises the magnetic attraction of the particles on a solid support. For example, patent documents U.S. Pat. Nos. 5,834,197 and 6,159,689, both of the same authors, describe methods for capturing and labeling a species, which consists of the attraction of particles having affinity for the species sought. The method comprises the magnetic attraction of said particles on a solid support by magnetic forces, and being immobilized, thus forcing the circulation of the sample and making it pass through the support. On one hand, it is evident that the number of favorable collisions for an antigen-antibody interaction will be smaller because the particles are fixed in a support, and part of the surface covered with antibodies is not accessible, and on the other hand, the exposed area is always the same and only a fraction of the area is actually available, such that the steric hindrance due to the initially captured bacteria limits the efficiency of new collisions very soon. This loss of effective area is not only due to the permanent contact with the support, but also to the aggregation of the particles, which is favored when they are retained on the support and are very close to one another, which increases this loss of efficiency even further. Furthermore, the same sample is repeatedly recirculated through the support with the retained particles; therefore there is no possibility of refreshing the sample in loads, but rather a single load of sample per recirculation is used up.
Document WO 02/101354 A2, which relates to kits and methods for the detection of microorganisms in a sample, also describes a method which comprises adhering capture antibodies specific for a marker of the microorganisms to a solid support; followed by adding second antibodies which may be conjugated to a molecule denoting the presence of the microorganisms, preferably by means of light which can be detected.
Document ES 2 208 121 A1 also relates to a method for the identification and quantification of analytes in which the antibodies and the antigens are immobilized, but instead of on a solid support as in WO 02/101354 A2, U.S. Pat. Nos. 5,834,197 and 6,159,689, on magnetic silica particles which are used as biosensors. The magnetic particles of the invention are iron oxide nanoparticles obtained by the Massart method, with a size of 5 to 30 nm, coated by a silica layer with a thickness of 30 to 100 nm.
The abstract of the document WO 2006/123781 A1 also relates to the use of magnetic silica particles in methods for recovering a microorganism from a sample, for which the sample is contacted with the particles absorbing it. The particles are characterized in that they have a diameter of 6 μm or less and their specific surface area is 50 m2/g or less.
Document US 2006/0211061 A1 relates to methods for the rapid detection of pathogenic microorganisms in a fluid by means of immunoassays. The method consists of binding a magnetic microparticle to a first epitope of the microorganism in a fluid by means of an antibody; using a magnetic field to separate the magnetic microparticle bound to the microorganism; binding a glucose molecule through a second antibody to the second epitope of the microorganism in question; and detecting the glucose in the sample to determine the presence and the concentration of the microorganism. The microparticles comprise microspheres of a superparamagnetic material coated with a polymer or protein, for example, albumin or avidin.
However, these methods have drawbacks which hinder their industrial application. These drawbacks include the following, among others:
1) The immobilization of the antibodies on the surface of the magnetic particles requires the presence in said surface of reactive groups, for example hydroxyl, amino or carboxyl groups. Once the antibodies have been bound to the surface by means of said reactive groups, there may be free groups which represent active sites to which other compounds present in the sample which can interfere in the antigen-antibody interaction, or in the composition of the developing reagents, or even the immobilized antibodies themselves the orientation of which to the external medium is altered, can also bind, making the interaction with the antigen and consequently the capture and recovery of the microorganism less likely.
2) The immunomagnetic particles collide with one another such that they can interact by means of weak bonds which can favor the formation of aggregates before the mixing with the sample, or after the mixing with the sample, an effect which depends on the concentration of the particle and on the contact time. This limits the possibility of reducing the limit of detection by increasing the amount of particle, and limits the useful life of the method based on using the particles.
3) The magnetic particles mixed with the complex sample can interact with some components which can favor the formation of aggregates, such that the interaction of the particle with the microorganism is less likely, and such that the efficiency of the magnetic retention is lower; consequently it makes the capture and recovery of the microorganism of interest less likely and the efficiency thereof decreases.
4) The quantitative recovery and handling of the immunomagnetic particles is not possible, fundamentally due to the previous drawback and mainly in large volumes of sample; consequently it is not possible to reduce the limit of detection by means of using large volumes of sample because there are variable losses of immunomagnetic particles and of complexes between the immunomagnetic particles and the microorganisms.
5) Some components present in the complex sample or in the solutions coming into contact with the particles in any or several of the separation steps can alter their coating, favoring the desorption of the blocking molecule, reactive groups being exposed in which other molecules which can be detrimental to the interaction of the microorganism with the immobilized antibody (capturing antibody), or said immobilized antibody, or the reading antibody, can be adsorbed.
6) The composition and concentration of different antigens which the microorganisms expose on their surface can change in response to changes in the environmental conditions (Albers U, Tiaden A, Spirig T, Al Alam D, Goyert S M, Gangloff S C, Hilbi H., “Expression of Legionella pneumophila paralogous lipid A biosynthesis genes under different growth conditions”, Microbiology, 153 (Pt 11):3817-29, 2007), and consequently the sensitivity and reproducibility of the determination of the microorganism can depend on the origin of the sample and its environmental conditions.
7) The microorganisms of interest which are captured by means of the immunomagnetic particles can have endogenous enzymes which interfere with the reading of the complexes which they form with said particles, and which cannot be separated and removed without altering the structural integrity of the captured microorganism. These interferences are dependent on the concentration of the captured microorganism, such that for high concentrations of the microorganism, said interferences can cause an underestimation of the amount of the microorganism in a quantitative or semi-quantitative determination, or cause a false negative in a qualitative determination. In particular, the non-obligate anaerobic (aerobic, facultative anaerobic, aerotolerant and microaerophilic) microorganisms, such as, among others, Escherichia coli, Staphylococcus, Legionella, Klebsiella, Bacillus, Salmonella, Campylobacter or Listeria, have an endogenous enzyme, catalase, which competes for the hydrogen peroxide added as a substrate of the peroxidase enzyme, usually conjugated to the reading antibody.
In recent years there has been an increasing demand for information related to the determination of analytes of a varied nature in complex samples, and in increasingly more varied areas, in a rapid, simple and sensitive manner. To overcome the difficulties of conventional methods which prevent meeting this demand, a great effort in the analytical instrumentation field has been directed to obtaining devices the use of which does not require professional supervision, the handling of which is simple and the cost of which is lower, capable of providing analytical information in a rapid, selective, sensitive, reliable and decentralized manner.
This demand has favored the development of biosensors as analysis alternatives to conventional analytical instrumentation, for the purpose of separating the analyte from the complex matrix in which it is located and measuring its presence or its concentration.
The preparation of biosensors based on using magnetic particles has opened new perspectives of applications to any analysis with solid supports, especially in automatic systems.
There are several patent documents which relates to apparatuses working as biosensors and serving for the detection of microorganisms. Thus, document ES 2 220 227 A1 relates to a method and apparatus for the detection of substances or analytes from the analysis of one or several samples. The invention relates to a robotic apparatus which can be handled by remote control and to a method which allows analyzing multiple natural samples. Said invention benefits from the technology of proteins and DNA microarrays. The apparatus comprises a series of operative modules, in which the samples are handled, processed and analyzed, and a series of control modules, which supervise the operation of said operative modules. The method for analyzing the sample comprises reacting said sample with a biosensor, washing the excess of unreacted sample and detecting the sample retained in the biosensor. Document WO 93/25909 A1 relates to an apparatus for the detection of the presence of analytes of interest in a sample, particularly biosensors, as well as to the method for detecting the presence of an analyte, and document U.S. Pat. No. 7,220,596 B2 relates to the detection of antigens which can be captured and detected from samples such as foods, for example, in approximately 30 minutes by using an apparatus and method including the passage of the sample through a module containing antibodies bound to particles. The flow of the sample through the modified particles is 0.2 to 1.2 L/minute. The antigens are thus captured by the antibodies and then the detection of the antibodies is carried out by fluorescence, chemiluminescence, or spectrometry techniques.
Therefore, a method which can be carried out in situ, for example, by means of a kit, which allows detecting and semi-quantifying a certain pathogenic microorganism in a minimum time, such as 1 hour, for example, to be able to immediately take the necessary measurements, is still necessary.
Therefore, the present invention proposes simple kits and processes for the rapid and sensitive determination of the presence of microorganisms in a wide range of samples of an environmental or food origin, as well as in biological fluids, by means of immunomagnetic particles in suspension, which overcomes the previous drawbacks, allowing obtaining the result of the in situ analysis, in a time less than or equal to one hour, without limitation of volume of the sample, and for concentrations of the microorganism of interest of the order of 1 cell per milliliter, and enables the industrial application thereof.